The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive, spore-forming bacterium traditionally characterized by parasporal crystalline protein inclusions. These inclusions often appear microscopically as distinctively shaped crystals. The proteins can be highly toxic to pests and specific in their toxic activity. Certain B.t. toxin genes have been isolated and sequenced, and recombinant DNA-based B.t. products have been produced and approved for use. In addition, with the use of genetic engineering techniques, new approaches for delivering B.t. toxins to agricultural environments are under development, including the use of plants genetically engineered with endotoxin genes for insect resistance and the use of stabilized intact microbial cells as B.t. toxin delivery vehicles (Gaertner, F. H., L. Kim [1988] TIBTECH 6:S4-S7; Beegle, C. C., T. Yamamoto, xe2x80x9cHistory of Bacillus thuringiensis Berliner research and development,xe2x80x9d Can. Ent. 124:587-616). Thus, isolated B.t. toxin genes are becoming commercially valuable.
Until the last fifteen years, commercial use of B.t. pesticides has been largely restricted to a narrow range of lepidopteran (caterpillar) pests. Preparations of the spores and crystals of B. thuringiensis subsp. kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example, B. thuringiensis var. kurstaki HD-1 produces a crystalline xcex4-endotoxin which is toxic to the larvae of a number of lepidopteran insects.
Investigators have now discovered B.t. pesticides with specificities for a much broader range of pests. For example, other species of B.t., namely israelensis and morrisoni (a.k.a. tenebrionis, a.k.a. B.t. M-7, a.k.a. B.t. san diego), have been used commercially to control insects of the orders Diptera and Coleoptera, respectively (Gaertner, F. H. [1989] xe2x80x9cCellular Delivery Systems for Insecticidal Proteins: Living and Non-Living Microorganisms,xe2x80x9d in Controlled Delivery of Crop Protection Agents, R. M. Wilkins, ed., Taylor and Francis, New York and London, 1990, pp. 245-255). See also Couch, T. L. (1980) xe2x80x9cMosquito Pathogenicity of Bacillus thuringiensis var. israelensis,xe2x80x9d Developments in Industrial Microbiology 22:61-76; and Beegle, C. C. (1978). xe2x80x9cUse of Entomogenous Bacteria in Agroecosystems,xe2x80x9d Developments in Industrial Microbiology 20:97-104. Krieg, A., A. M. Huger, G. A. Langenbruch, W. Schnetter (1983) Z. ang. Ent. 96:500-508 describe Bacillus thuringiensis var. tenebrionis, which is reportedly active against two beetles in the order Coleoptera. These are the Colorado potato beetle, Leptinotarsa decemlineata, and Agelastica alni. 
More recently, new subspecies of B.t. have been identified, and genes responsible for active xcex4-endotoxin proteins have been isolated (Hxc3x6fte, H., H. R. Whiteley [1989] Microbiological Reviews 52(2):242-255). Hxc3x6fte and Whiteley classified B.t. crystal protein genes into four major classes. The classes were CryI (Lepidoptera-specific), CryII (Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), and CryIV (Diptera-specific). The discovery of strains specifically toxic to other pests has been reported (Feitelson, J. S., J. Payne, L. Kim [1992] Bio/Technology 10:271-275). CryV has been proposed to designate a class of toxin genes that are nematode-specific. Lambert et al. (Lambert, B., L. Buysse, C. Decock, S. Jansens, C. Piens, B. Saey, J. Seurinck, K. van Audenhove, J. Van Rie, A. Van Vliet, M. Peferoen [1996] Appl. Environ. Microbiol. 62(1):80-86) describe the characterization of a Cry9 toxin active against lepidopterans. Published PCT applications WO 94/05771 and WO 94/24264 also describe B.t. isolates active against lepidopteran pests. Gleave et al. ([1991] JGM 138:55-62), Shevelev et al. ([1993] FEBS Lett. 336:79-82), and Smulevitch et al. ([1991] FEBS Lett. 293:25-26) also describe B.t. toxins. Many other classes of B.t. genes have now been identified.
The cloning and expression of a B.t. crystal protein gene in Escherichia coli has been described in the published literature (Schnepf, H. E., H. R. Whiteley [1981] Proc. Natl. Acad. Sci. USA 78:2893-2897). U.S. Pat. No. 4,448,885 and U.S. Pat. No. 4,467,036 both disclose the expression of B.t. crystal protein in E. coli. U.S. Pat. Nos. 4,990,332; 5,039,523; 5,126,133; 5,164,180; and 5,169,629 are among those which disclose B.t. toxins having activity against lepidopterans. PCT application WO 96/05314 discloses PS86W1, PS86V1, and other B.t. isolates active against lepidopteran pests. The PCT patent applications published as WO 94/24264 and WO 94/05771 describe B.t. isolates and toxins active against lepidopteran pests. B.t. proteins with activity against members of the family Noctuidae are described by Lambert et al., supra. U.S. Pat. Nos. 4,797,276 and 4,853,331 disclose B. thuringiensis strain tenebrionis which can be used to control coleopteran pests in various environments. U.S. Pat. No. 4,918,006 discloses B.t. toxins having activity against dipterans. U.S. Pat. No. 5,151,363 and U.S. Pat. No. 4,948,734 disclose certain isolates of B.t. which have activity against nematodes. Other U.S. patents which disclose activity against nematodes include 5,093,120; 5,236,843; 5,262,399; 5,270,448; 5,281,530; 5,322,932; 5,350,577; 5,426,049; and 5,439,881.
A cry2Aa gene from HD263 kurstaki is disclosed by Donovan et al. in 264 JBC 4740 (1989). Another cry2Aa gene and a cry2Ab gene, from HD1 kurstaki, are disclosed by Widner and Whiteley, 171 J. Bac. 965-974 (1989). Another cry2Ab gene from HD1 kurstaki is disclosed by Dankocsik et al. in 4 Mol. Micro. 2087-2094 (1990). A cry2Ac gene from B.t.S-1 (shanghai) is disclosed by Wu et al. in 81 FEMS 31-36 (1991).
An isolate known as PS192M4 is disclosed in U.S. Pat. No. 5,273,746 as having activity against lice.
The PS86I2 isolate is disclosed in U.S. Pat. No. 5,686,069 as having activity against lepidopterans. PS91C2 is exemplified therein as producing a CryIF(b)-type of lepidopteran-active toxin, the sequence of which is disclosed therein.
Sequence information for a lepidopteran-active toxin from HD525 and the sequence of a lepidopteran-active toxin from HD573 are disclosed in WO 98/00546. Those toxins are not Cry2-type toxins.
As a result of extensive research and investment of resources, other patents have issued for new B.t. isolates and new uses of B.t. isolates. See Feitelson et al., supra, for a review. However, the discovery of new B.t. isolates and new uses of known B.t. isolates remains an empirical, unpredictable art. U.S. Pat. No. 5,506,099 describes methods for identifying unknown B.t. isolates. U.S. Pat. No.. 5,204,237 describes specific and universal probes for the isolation of B.t. toxin genes. These patents, however, do not describe the probes and primers of the subject invention.
The subject invention concerns materials and methods useful in the control of non-mammalian pests and, particularly, plant pests. In a specific embodiment, the subject invention provides new toxins useful for the control of lepidopterans. A preferred embodiment of the subject invention further provides nucleotide sequences which encode the novel lepidopteran-active toxins of the subject invention.
The subject invention further provides nucleotide sequences and methods useful in the identification and characterization of novel genes which encode pesticidal toxins. In one embodiment, the subject invention concerns unique nucleotide sequences which are useful as primers in PCR techniques. The primers produce characteristic gene fragments which can be used in the identification and isolation of novel toxin genes. A further aspect of the subject invention is the use of the disclosed nucleotide sequences as probes to detect genes encoding B.t. toxins which are active against lepidopterans.
Further aspects of the subject invention include other novel genes and toxins identified using the methods and nucleotide sequences disclosed herein, in addition to the novel genes and toxins specifically disclosed herein. The genes thus identified encode toxins active against lepidopterans. Similarly, the isolates capable of producing these toxins have activity against these pests. Thus, the subject invention further provides new Bacillus thuringiensis isolates having pesticidal activities which are found with the primers and probes according to the subject invention.
In one embodiment of the subject invention, B.t. isolates can be cultivated under conditions resulting in high multiplication of the microbe. After treating the microbe to provide single-stranded genomic nucleic acid, the DNA can be contacted with the primers of the invention and subjected to PCR amplification. Characteristic fragments of toxin-encoding genes are amplified by the procedure, thus identifying the presence of the toxin-encoding gene(s).
In a preferred embodiment, the subject invention concerns plants cells transformed with at least one polynucleotide sequence of the subject invention such that the transformed plant cells express pesticidal toxins in tissues consumed by the target pests. Such transformation of plants can be accomplished using techniques well known to those skilled in the art and would typically involve modification of the gene to optimize expression of the toxin in plants. In addition, the toxins of the subject invention may be chimeric toxins produced by combining portions of multiple toxins.
As an alternative to the transformation of plants, the B.t. isolates and toxins of the subject invention, or recombinant microbes expressing the toxins described herein, can be used to control pests. In this regard, the invention includes the treatment of substantially intact B.t. cells, and/or recombinant cells containing the expressed toxins of the invention, treated to prolong the pesticidal activity when the substantially intact cells are applied to the environment of a target pest. The treated cell acts as a protective coating for the pesticidal toxin. The toxin becomes active upon ingestion by a target insect.
SEQ ID NO. 1 is a forward primer useful according to the subject invention.
SEQ ID NO. 2 is a reverse primer useful according to the subject invention.
SEQ ID NO. 3 is a nucleotide sequence which encodes the 192M4 toxin.
SEQ ID NO. 4 is the predicted amino acid sequence of the 192M4 toxin.
SEQ ID NO. 5 is a nucleotide sequence which encodes the HD573 toxin.
SEQ ID NO. 6 is the predicted amino acid sequence of the HD573 toxin.
SEQ ID NO. 7 is a nucleotide sequence which encodes the HD525 toxin.
SEQ ID NO. 8 is the predicted amino acid sequence of the HD525 toxin.
SEQ ID NO. 9 is a nucleotide sequence which encodes the 86I2 toxin.
SEQ ID NO. 10 is the predicted amino acid sequence of the 86I2 toxin.
The subject invention concerns materials and methods for the control of non-mammalian pests. In specific embodiments, the subject invention pertains to new Bacillus thuringiensis toxins, and genes encoding toxins, which have activity against lepidopterans. The subject invention concerns not only the polynucleotide sequences which encode these toxins, but also the use of these polynucleotide sequences to produce recombinant hosts which express the toxins. The subject invention further concerns novel nucleotide sequences that are useful as primers and probes for Bacillus thuringiensis (B.t.) genes that encode pesticidal toxins, especially lepidopteran-active toxins. The subject invention still further concerns novel methods for identifying and characterizing B.t. isolates, toxins, and genes with useful properties.
The new toxins and polynucleotide sequences provided here are defined according to several parameters. One critical characteristic of the toxins described herein is pesticidal activity. In a specific embodiment, these toxins have activity against lepidopteran pests. The toxins and genes of the subject invention can be further defined by their amino acid and nucleotide sequences. The sequences of the molecules can be defined in terms of homology or identity to certain exemplified sequences as well as in terms of the ability to hybridize with, or be amplified by, certain exemplified probes and primers. The toxins provided herein can also be identified based on their immunoreactivity with certain antibodies.
Methods have been developed for making useful chimeric toxins by combining portions of B.t. crystal proteins. The portions which are combined need not, themselves, be pesticidal so long as the combination of portions creates a chimeric protein which is pesticidal. This can be done using restriction enzymes, as described in, for example, European Patent 0 228 838; Ge, A. Z., N. L. Shivarova, D. H. Dean (1989) Proc. Natl. Acad. Sci. USA 86:4037-4041; Ge, A. Z., D. Rivers, R. Milne, D. H. Dean (1991) J. Biol. Chem. 266:17954-17958; Schnepf, H. E., K. Tomczak, J. P. Ortega, H. R. Whiteley (1990) J. Biol. Chem. 265:20923-20930; Honee, G., D. Convents, J. Van Rie, S. Jansens, M. Peferoen, B. Visser (1991) Mol. Microbiol. 5:2799-2806. Alternatively, recombination using cellular recombination mechanisms can be used to achieve similar results. See, for example, Caramori, T., A. M. Albertini, A. Galizzi (1991) Gene 98:37-44; Widner, W. R.; H. R. Whiteley (1990) J. Bacteriol. 172:2826-2832; Bosch, D., B. Schipper, H. van der Kliej, R. A. de Maagd, W. J. Stickema (1994) Biotechnology 12:915-918. A number of other methods are known in the art by which such chimeric DNAs can be made. The subject invention is meant to include chimeric proteins that utilize the novel sequences identified in the subject application.
With the teachings provided herein, one skilled in the art could readily produce and use the various toxins and polynucleotide sequences described herein.
B.t. isolates useful according to the subject invention have been deposited in the permanent collection of the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 North University Street, Peoria, Ill. 61604, USA. The culture repository numbers of the B.t. strains are as follows:
Cultures have been deposited under conditions that assure that access to the cultures is available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. 122. The deposits will be available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.
Further, the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, i.e., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the culture(s). The depositor acknowledges the duty to replace the deposit(s) should the depository be unable to furnish a sample when requested, due to the condition of a deposit. All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.
The isolates HD525 and HD573 are available from the USDA-ARS NRRL Culture Collection, Peoria, Ill.
Following is a table which provides characteristics of certain isolates useful according to the subject invention.
Genes and toxins. The genes and toxins useful according to the subject invention include not only the full length sequences but also fragments of these sequences, variants, mutants, and fusion proteins which retain the characteristic pesticidal activity of the novel toxins specifically exemplified herein. Chimeric genes and toxins, produced by combining portions from more than one B.t. toxin or gene, may also be utilized according to the teachings of the subject invention. As used herein, the terms xe2x80x9cvariantsxe2x80x9d or xe2x80x9cvariationsxe2x80x9d of genes refer to nucleotide sequences which encode the same toxins or which encode equivalent toxins having pesticidal activity. As used herein, the term xe2x80x9cequivalent toxinsxe2x80x9d refers to toxins having the same or essentially the same biological activity against the target pests as the exemplified toxins.
It should be apparent to a person skilled in this art that genes encoding active toxins can be identified and obtained through several means. The specific genes exemplified herein may be obtained from the isolates deposited at a culture depository as described above. These genes, or portions or variants thereof, may also be constructed synthetically, for example, by use of a gene synthesizer. Variations of genes maybe readily constructed using standard techniques for making point mutations. Also, fragments of these genes can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Also, genes which encode active fragments may be obtained using a variety of restriction enzymes. Proteases may be used to directly obtain active fragments of these toxins.
Equivalent toxins and/or genes encoding these equivalent toxins can be derived from B.t. isolates and/or DNA libraries using the teachings provided herein. There are a number of methods for obtaining the pesticidal toxins of the instant invention. For example, antibodies to the pesticidal toxins disclosed and claimed herein can be used to identify and isolate other toxins from a mixture of proteins. Specifically, antibodies may be raised to the portions of the toxins which are most constant and most distinct from other B.t. toxins.
Fragments and equivalents which retain the pesticidal activity of the exemplified toxins would be within the scope of the subject invention. Also, because of the redundancy of the genetic code, a variety of different DNA sequences can encode the amino acid sequences disclosed herein. It is well within the skill of a person trained in the art to create these alternative DNA sequences encoding the same, or essentially the same, toxins. These variant DNA sequences are within the scope of the subject invention. As used herein, reference to xe2x80x9cessentially the samexe2x80x9d sequence refers to sequences which have amino acid substitutions, deletions, additions, or insertions which do not materially affect pesticidal activity. Fragments retaining pesticidal activity are also included in this definition.
Certain toxins of the subject invention have been specifically exemplified herein. Since these toxins are merely exemplary of the toxins of the subject invention, it should be readily apparent that the subject invention also relates to variants or equivalents of novel genes and toxins having the same or similar pesticidal activity of the exemplified novel toxins. Equivalent toxins will have amino acid homology with a novel exemplified toxin. These equivalent genes and toxins will typically have greater than 60% identity with the sequences specifically exemplified herein; preferably, there will be more than 75% identity, more preferably greater than 80%, most preferably greater than 90%, and the identity can be greater than 95%. The amino acid homology will be highest in critical regions of the toxin which account for biological activity or are involved in the determination of three-dimensional configuration which ultimately is responsible for the biological activity. In this regard, conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound. Table 4 provides a listing of examples of amino acids belonging to each class.
In some instances, non-conservative substitutions can also be made. The critical factor is that these substitutions must not significantly detract from the biological activity of the toxin.
The toxins of the subject invention can also be characterized in terms of the shape and location of toxin inclusions, which are described above. Although novel crystal proteins are specifically exemplified herein, isolates for use according to the subject invention can be grown under conditions that facilitate the secretion of toxins. Thus, the supernatant from these cultures can be used to obtain toxins according to the subject invention. Thus, the subject invention is not limited to crystal proteins; useful soluble proteins are also contemplated.
As used herein, reference to xe2x80x9cisolatedxe2x80x9d polynucleotides and/or xe2x80x9cpurifiedxe2x80x9d toxins refers to these molecules when they are not associated with the other molecules with which they would be found in nature. Thus, reference to xe2x80x9cisolated and purifiedxe2x80x9d signifies the involvement of the xe2x80x9chand of manxe2x80x9d as described herein. Chimeric toxins and genes also involve the xe2x80x9chand of man.xe2x80x9d
The use of oligonucleotide probes provides a method for identifying the toxins and genes of the subject invention, and additional novel genes and toxins. Probes provide a rapid method for identifying toxin-encoding genes. The nucleotide segments which are used as probes according to the invention can be synthesized using a DNA synthesizer and standard procedures, for example.
Recombinant hosts. The toxin-encoding genes of the subject invention can be introduced into a wide variety of microbial or plant hosts. Expression of the toxin gene results, directly or indirectly, in the production and maintenance of the pesticide. With suitable microbial hosts, e.g., Pseudomonas, the microbes can be applied to the situs of the pest, where they will proliferate and be ingested. The result is a control of the pest. Alternatively, the microbe hosting the toxin gene can be killed and treated under conditions that prolong the activity of the toxin and stabilize the cell. The treated cell, which retains the toxic activity, then can be applied to the environment of the target pest.
A wide variety of methods are available for introducing a B.t. gene encoding a toxin into a microorganism host under conditions which allow for stable maintenance and expression of the gene. These methods are well known to those skilled in the art and are described, for example, in U.S. Pat. No. 5,135,867, which is incorporated herein by reference.
Alternatively, a plant transformed to express a toxin of the subject invention can be used to contact the target pest with the toxin. Synthetic genes which are functionally equivalent to the novel toxins of the subject invention can also be used to transform hosts. Methods for the production of synthetic genes can be found in, for example, U.S. Pat. No. 5,380,831.
Treatment of cells. As mentioned above, B.t. or recombinant cells expressing a B.t. toxin can be treated to prolong the toxin activity and stabilize the cell. The pesticide microcapsule that is formed comprises the B.t. toxin within a cellular structure that has been stabilized and will protect the toxin when the microcapsule is applied to the environment of the target pest. Methods for treatment of microbial cells are disclosed in U.S. Pat. Nos. 4,695,455 and 4,695,462, which are incorporated herein by reference.
Growth of cells. The cellular host containing the B.t. insecticidal gene may be grown in any convenient nutrient medium, where the DNA construct provides a selective advantage, providing for a selective medium so that substantially all or all of the cells retain the B.t. gene. These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be treated prior to harvesting.
The B.t. cells of the invention can be cultured using standard art media and fermentation techniques. Upon completion of the fermentation cycle the bacteria can be harvested by first separating the B.t. spores and crystals from the fermentation broth by means well known in the art. Any B.t. spores and crystals can be recovered employing well-known techniques and used as a conventional xcex4-endotoxin B.t. preparation. For example, the spores and crystals can be formulated into a wettable powder, liquid concentrate, granules or other formulations by the addition of surfactants, dispersants, inert carriers, and other components to facilitate handling and application for particular target pests. These formulations and application procedures are all well known in the art. Alternately, the supernatant from the fermentation process can be used to obtain toxins according to the present invention. Soluble, secreted toxins are then isolated and purified employing well-known techniques.
Methods and formulations for control of pests. Control of lepidopterans using the isolates, toxins, and genes of the subject invention can be accomplished by a variety of methods known to those skilled in the art. These methods include, for example, the application of B.t. isolates to the pests (or their location), the application of recombinant microbes to the pests (or their locations), and the transformation of plants with genes which encode the pesticidal toxins of the subject invention. Recombinant microbes may be, for example, a B.t., E. coli, or Pseudomonas. Transformations can be made by those skilled in the art using standard techniques. Materials necessary for these transformations are disclosed herein or are otherwise readily available to the skilled artisan.
Formulated bait granules containing an attractant and toxins of the B.t. isolates, or recombinant microbes comprising the genes obtainable from the B.t. isolates disclosed herein, can be applied to the soil. Formulated product can also be applied as a seed-coating or root treatment or total plant treatment at later stages of the crop cycle. Plant and soil treatments of B.t. cells may be employed as liquids, wettable powders, granules, or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like).
As would be appreciated by a person skilled in the art, the pesticidal concentration will vary widely depending upon the nature of the particular formulation, particularly whether it is a concentrate or to be used directly. The pesticide will be present in at least about 1% by weight and may be 100% by weight. The dry formulations will have from about 1-95% by weight of the pesticide while the liquid formulations will generally be from about 1-60% by weight of the solids in the liquid phase. The formulations will generally have from about 102 to about 104 cells/mg. These formulations that contain cells will be administered at about 50 mg (liquid or dry) to 1 kg or more per hectare.
The formulations can be applied to the environment of the pest, e.g., soil and foliage, by spraying, dusting, sprinkling, or the like.
Mutants. Mutants of novel isolates obtainable according to the invention can be made by procedures well known in the art. For example, an asporogenous mutant can be obtained through ethylmethane sulfonate (EMS) mutagenesis of an isolate. The mutants can be made using ultraviolet light and nitrosoguanidine by procedures well known in the art.
Polynucleotide probes. Hybridization may be used to test whether two pieces of DNA are complementary in their base sequences. It is this hybridization mechanism which facilitates the use of probes of the subject invention to readily detect and characterize DNA sequences of interest. The probes may be RNA or DNA. The probe will normally have at least about 10 bases, more usually at least about 18 bases, and may have up to about 50 bases or more, usually not having more than about 200 bases if the probe is made synthetically. However, longer probes can readily be utilized, and such probes can be, for example, several kilobases in length. The probes may be labeled utilizing techniques which are well known to those skilled in this art.
One approach for the use of the subject invention as probes entails first identifying by Southern blot analysis of a gene bank of the B.t. isolate all DNA segments homologous with the disclosed nucleotide sequences. Thus, it is possible, without the aid of biological analysis, to know in advance the probable activity of many new B.t. isolates, and of the individual endotoxin gene products expressed by a given B.t. isolate. Such a probe analysis provides a rapid method for identifying potentially commercially valuable insecticidal endotoxin genes within the multifarious subspecies of B.t.
The particular hybridization technique is not essential to the subject invention. As improvements are made in hybridization techniques, they can be readily applied.
The nucleotide segments of the subject invention which are used as probes can be synthesized by use of DNA synthesizers using standard procedures. In the use of the nucleotide segments as probes, the particular probe is labeled with any suitable label known to those skilled in the art, including radioactive and non-radioactive labels.
Various degrees of stringency of hybridization can be employed. The more severe the conditions, the greater the complementarity that is required for duplex formation. Severity can be controlled by temperature, probe concentration, probe length, ionic strength, time, and the like. Preferably, hybridization is conducted under stringent conditions by techniques well known in the art, as described, for example, in Keller, G. H., M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y., pp. 169-170.
As used herein xe2x80x9cstringentxe2x80x9d conditions for hybridization refers to conditions which achieve the same, or about the same, degree of specificity of hybridization as the conditions employed by the current applicants. Specifically, hybridization of immobilized DNA on Southern blots with 32P-labeled gene-specific probes was performed by standard methods (Maniatis et al.). In general, hybridization and subsequent washes were carried out under stringent conditions that allowed for detection of target sequences with homology to the exemplified toxin genes. For double-stranded DNA gene probes, hybridization was carried out overnight at 20-25xc2x0 C. below the melting temperature (Tm) of the DNA hybrid in 6xc3x97SSPE, 5xc3x97Denhardt""s solution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature is described by the following formula (Beltz, G. A., K. A. Jacobs, T. H. Eickbush, P. T. Cherbas, and F. C. Kafatos [1983] Methods of Enzymology, R. Wu, L. Grossman and K. Moldave [eds.] Academic Press, New York 100:266-285):
Tm=81.5xc2x0 C.+16.6 Log[Na+]+0.41(% G+C)xe2x88x920.61(% formamide)xe2x88x92600/length of duplex in base pairs.
Washes are typically carried out as follows:
(1) Twice at room temperature for 15 minutes in 1xc3x97SSPE, 0.1% SDS (low stringency wash).
(2) Once at Tmxe2x80x9420xc2x0 C. for 15 minutes in 0.2xc3x97SSPE, 0.1% SDS (moderate stringency wash).
For oligonucleotide probes, hybridization was carried out overnight at 10-20xc2x0 C. below the melting temperature (Tm) of the hybrid in 6xc3x97SSPE, 5xc3x97Denhardt""s solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm for oligonucleotide probes was determined by the following formula:
Tm (xc2x0 C.)=2(number T/A base pairs)+4(number G/C base pairs) (Suggs, S. V., T. Miyake, E. H. Kawashime, M. J. Johnson, K. Itakura, and R. B. Wallace [1981] ICN-UCLA Symp. Dev. Biol. Using Purified Genes, D. D. Brown [ed.], Academic Press, New York, 23:683-693).
Washes were typically carried out as follows:
(1) Twice at room temperature for 15 minutes 1xc3x97SSPE, 0.1% SDS (low stringency wash).
(2) Once at the hybridization temperature for 15 minutes in 1xc3x97SSPE, 0.1% SDS (moderate stringency wash).
PCR technology. The DNA sequences of the subject invention can be used as primers for PCR amplification. In performing PCR amplification, a certain degree of mismatch can be tolerated between primer and template. Therefore, mutations, deletions, and insertions (especially additions of nucleotides to the 5xe2x80x2 end) of the exemplified primers fall within the scope of the subject invention. Mutations, insertions and deletions can be produced in a given primer by methods known to an ordinarily skilled artisan.
Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.